Pathology - Theses
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ItemInvestigating the mechanistic link between neuroinflammation and biometal homeostasis in neurodegenerative diseases( 2016)Neuroinflammation and biometal dyshomeostasis are two pathogenic features underlying a number of neurodegenerative diseases, however the mechanistic link between these two pathways has yet to be delineated. This study examined the hypothesis that impaired biometal homeostasis is associated with neuroinflammatory changes. To test this hypothesis I aimed to investigate the effects of key biometals on inflammatory processes in cultured microglia, and in turn, investigate how inflammatory activation of microglia affects homeostasis of biometals. These relationships were further examined in vivo to determine the effects of the type 1 interferon (IFN) pathway on biometal homeostasis in the CNS. In my in vitro study, primary murine microglial cultures were treated for 24h with maximal sub-toxic doses of biometals, delivered as ferric ammonium chloride (FAC), ZnCl2 and CuCl2 and the biometal chelators, diamsar or N,N,N_,N_-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) with and without concurrent interferon-_ (IFN_) and tumour necrosis factor-_ (TNF_) stimulation. Non-stimulated and IFN_/TNF_ stimulated microglia served as negative and positive controls for inflammatory activated microglia, respectively. I measured the levels of a number of key inflammatory cytokines to assess microglial inflammatory response to biometal and biometal chelator treatments. I found that FAC and CuCl2 treatment, significantly induced Fe and Cu uptake respectively, in both non-stimulated and stimulated microglia and that all biometal treatments, significantly reduced the expression of MCP-1 in stimulated and non-stimulated microglia, indicative of an anti-inflammatory role. In contrast, FAC treatment also induced TNF_ mRNA expression in these cultures, suggesting Fe may play a dual role in neuroinflammation. In addition, to investigate how inflammatory activation of microglia affects biometal homeostasis, the gene expression of the metal-binding protein, metallothionein-1 (MT-1) and the biometal transporter, ZRT/IRT-like transporter protein (Zip7) were also measured. I also found that IFN_/TNF_ stimulation inhibited Fe-induced MT-1 and Zip7 expression in microglia. These findings demonstrate that sub-toxic levels of key biometals have multiple modulatory actions on cultured microglia, with both inhibitory and stimulatory effects on cytokines. These changes may be associated with induction or inhibition of major metal response proteins, such as MT-1 and transporters. To examine the effects of the type 1 IFN pathway on biometal homeostasis in the CNS, I performed a spatio-temporal analysis of Fe, Zn, Cu and Mn levels in the CNS of interferon _ receptor-1 (Ifnar1) knock-out (-/-) mice and wild type (WT) mice at 6 and 10 months of age using ICP-MS analysis. A subset of 6-month-old Ifnar1-/- mice was also stimulated with lipopolysaccharide (LPS) treatment for 6h to determine the effects of Ifnar1-/- on biometals homeostasis under inflammatory conditions. I found reduced Cu and Mn levels in the cerebellum of aged (10-month-old) Ifnar1-/- mice, however, expression of key Cu and Mn transporter and regulatory proteins remained unchanged. I also found no significant alterations to biometals between WT and Ifnar1-/- mice at 6-month of age, however, when mice were challenged with LPS, I found a significant decrease in Fe levels in the cerebellum and cerebrum of WT mice and a significant decrease in Zn levels in the cerebrum of Ifnar1-/- mice compared to naïve mice of their respective genotypes. A significant increase and an upward trend in transferrin receptor1 (TfR1) levels in the cerebrum of LPS-challenged and naïve Ifnar1-/- mice, respectively was also observed. These data demonstrate that the type 1 IFN pathway is involved in the regulation of CNS biometal homoeostasis. The studies provide further evidence to support a major role for biometals in neuroinflammatory pathways, with important implications for neurodegenerative disease in which brain biometal homeostasis is altered.
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ItemThe mechanism of action of CuII(atsm) for the treatment of amyotrophic lateral sclerosis( 2015)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the progressive loss of motor neurons in the spinal cord, motor cortex and brain stem leading to complete paralysis and death, usually within 2-3 years of diagnosis. There is currently no cure for ALS and the only approved therapeutic is riluzole. However, its clinical efficacy is marginal with an average extension in survival of 3 months. A subset of ALS cases (~10%) can be attributed to genetically inherited mutations in a number of different genes (familial ALS). Mutations in the gene for Cu,Zn superoxide dismutase (SOD1) – an antioxidant enzyme – were the first to be identified. These mutations lead to a toxic gain of function but the exact nature of this toxicity remains largely unknown. There is evidence to suggest that mutations may cause incorrect metallation of SOD1 leading to aberrant catalytic chemistry and misfolding. Over-expression of the mutant forms of the human protein in mice gives rise to a phenotype that recapitulates many of the symptoms of the human condition including progressive paralysis and premature death. The PET imaging agent, diacetyl-bis(4-methylthiosemicarbazonato)Cu(II) [CuII(atsm)] has been shown to have therapeutic potential in one of these models – SOD1G93A mice. In addition, CuII(atsm) has also been shown to be protective in multiple models of Parkinson's disease. The purpose of this thesis was to further characterise the therapeutic potential of CuII(atsm) in a second model of ALS and to determine if its therapeutic mechanism involves modulation of Cu bioavailability in disease affected tissue. CuII(atsm) was shown to have similar therapeutic potential in the SOD1G37R model as in the SOD1G93A model. Survival extension and improvement in locomotor symptoms were dependent on the dose administered with the highest dose administered proving to be the most effective. No apparent therapeutic ceiling was reached. CuII(atsm) was also co-administered with riluzole with no apparent additive or detrimental effects. When administered alone, riluzole was not as effective at attenuating symptoms and survival as CuII(atsm). Additionally, CuII(atsm) was therapeutic even when given post-onset of a locomotor deficit. Even though severity of disease symptoms in these mice is dependent on mutant SOD1 expression levels, treatment with CuII(atsm) was shown to paradoxically increase the concentration of mutant SOD1 in the spinal cord of these mice. This was due to an increase in fully metallated holo SOD1 – the stable, non-toxic form of the enzyme. The holo SOD1 pool was increased by incorporation of Cu from CuII(atsm) into the Cu-deficient, Zn-containing SOD1 pool. Several other proteins also incorporated Cu from CuII(atsm) however, not all detectable cuproproteins were targets of CuII(atsm)-mediated Cu delivery. Preliminary results suggest that the cuproprotein targets of CuII(atsm) are involved in oxidative stress, metal homeostasis and Cu delivery to SOD1, potentially inhibiting the toxic action of metal-deficient SOD1 on mitochondria. The clinical and pathological similarities between familial and sporadic ALS suggest that similar pathological processes occur in both forms of the disease. There is evidence to suggest that SOD1 can cause disease in the absence of mutations and there is ample evidence implicating mitochondrial dysfunction in sporadic ALS as well as familial ALS. CuII(atsm) is therefore a promising therapeutic for the treatment of ALS and the results presented and mechanism proposed in this thesis position CuII(atsm) as an excellent candidate for translation into human clinical trials.